• What is R?
• Brief Intro To RStudio & R Concepts
• R Objects
• The R Workspace
• R Packages
• Getting Data Into R
• Basic Plotting

• A fully functional programming language!
• Originally developed @ Bell Labs for interactive data analysis
  • Was called S back then (~1976)
• Open sourced in 1993 under the name R
  • Designed to be "not unlike S"
• Strong emphasis on statistics
• Excellent for data visualization
• Gaining popularity in many circles

• This short tutorial can't cover the vast R universe
  • (or even come close :))
• But… R has vibrant community supporting it!
• You can find endless tutorials, books, presentations, etc. on just about any aspect of R you want – from beginner topics to advanced matters.
• Goal for today:

• RStudio provides many online learning resources
• And even more helpful links buried in an RStudio wiki page
• Code School has an excellent tutorial to help you get acquainted with R
• StackOverflow is a great place to find answers to R programming questions
• Google Developers intro to R video tutorials
• Advanced R by Hadley Wickham
  • The first few sections are excellent for beginners
  • The rest of the book is essential for a deep understanding of R

• The R console can be used as a calculator
• Enter the following commands into your R console to see it in action:

2 + 2
5 * 4
2^3

• It is also smart enough to understand order of operations:

2 + 3 * 4/(5 + 3) * 15/2^2 + 3 * 4^2

> y
Error: object 'y' not found

You can name your variables anything you want, but there are a few rules:

1. Names may contain letters, numbers or periods, and the underscore character
2. Names must start with a letter or period.
3. v.one and v_one are valid names but v one is not (because it includes a space).

More information on general R programming style can be found here.

1. Vectors
2. Matricies/Arrays
3. Data Frames
4. Lists

• Data frames are the R data structure you will deal with most frequently
• R's data import/export functions will return these by default
• They can also be created using the data.frame() function
• Here's a quick example creating some dummy experimental data:

> data.frame(group = rep(c('trt', 'ctrl'), each=4), 
            response = rnorm(8))
   group    response
1    trt  0.01231700
2    trt  0.55028627
3    trt -0.24411263
4    trt  0.35996580
5    trt  0.89960231
6   ctrl -0.76686861
7   ctrl  0.03573818
8   ctrl  1.69159332

• R provides users with many built in functions
  • These allow users to perform a range of tasks
  • Computing summary statistics, to fitting linear models, to generating random variables.
• There are lots of good lists of functions out there
• In most cases, R functions have been given logical names.
• Ex: the mean function is used to find the average of a vector of numbers:

> my_data <- c(10, 22, 44, 55, 14, 66)
> mean(my_data)
[1] 35.16667

And, if all else fails, try Googling to see if the function you seek exists!!

The R workspace can be thought of as a container holding all of the objects you've created duing your R session. You can print a list of all of the objects in your current workspace using the ls() function. If we start a new R session, our workspace will be empty:

> ls()
character(0)

And we'll be able to see some objects if we add them:

> x <- 20
> y <- 30
> z <- x + y
> df <- data.frame(nums = 1:10, b = letters[1:10])
> ls()
[1] "df" "x"  "y"  "z" 

• save.image: saves a snapshot of entire workspace to a file
  • Typical usage: save.image('my-work.RData')
• save: saves a snapshot of a few specified objects to a file.
  • Useful if you only want to save one or 2 things (like a modified data set).
  • To save, df, the data.frame we created in the last section:
  • save(df, file = 'my-dataframe.RData')
• load: loads your saved *.RData files back in to R
  • To load, df, the data.frame we just saved:
  • load('my-dataframe.RData')

• There are two main ways to clear your workspace in R/RStudio
  1. using the rm() function to remove objects and
  2. using the Clear button in RStudio.
• rm() lets you remove objects from your R session when they aren't needed
• Example of the usage:

> ls() # start in an empty workspace
character(0)
> y <- 1
> z <- 1
> ls() # can see the two objects we created
[1] "y" "z"
> rm(y) # remove y
> ls()  
[1] "z"

You can remove all objects in your workspace by using ls() to generate a vector of all the objects that have been created, and passing that to the rm() function:

rm(list = ls())

You can also use a button in RStudio's Environment panel to remove all of the objects in your workspace. RStudio will prompt you asking if you are sure you want to go through with deleting all objects, choosing Yes will permanently delete all objects in the workspace.

• Packages are what make R so great.
• Packages allow R users to develop and share new code to extend the language
  • e.g. implementing a new statistical model for analyzing microarrays, creating an awesome new graph to show copy number alterations in tumor samples, etc.
• R has become the lingua franca for statisticians developing new statistical techniques
• Has also been widely adopted in the bioinformatics community (Bioconductor)

Most generic R packages are hosted on the Comprehensive R Archive Network CRAN. To install one of these packages, you would use install.packages("packagename"). You only need to install a package once, then load it each time using library(packagename). Here's how one would install and load the ggplot2 package.

# Install only once.
install.packages("ggplot2")

# Load the package every time you want to use it.
library(ggplot2)

Now that we're feeling a bit more comfortable with the R environment, we'll explore how we can import our own experimental data into R.

Before going any further, please download the zip file below and extract its contents somewhere on your computer.

• data_dl.zip

• Easiest way to store data for import into R is in flat text files.
• Can import from other data stores (Excel sheet, database, etc)
  • But that is out of the scope of this tutorial
• Flat text files containing data typically have a column separator (delimiter)
  • think: tab, semicolon, space
• Two easy ones to export from Excel are (see handout for examples):
  • Tab-Delimited Files
  • Comma-Delimited Files

• Just saw RStudio generate a read.delim function to import our .tsv data frame
• read.delim is a special alternative to R's more general read.table function (?read.table for details)
• read.csv is another special alternative to read.table
• read.csv & read.delim have

• Oftentimes, analysts will want to work with only a subset of their data at one time
• A definitive guide on subsetting in R can be found here
• There are many ways to subset data in R, but we'll touch on a couple important ones

> gene.exprs.long[1:5, ] # print the first 5 rows
        ID   Group  Gene Expression
1 Sample 1 Group 1 Gene1   9.695228
2 Sample 2 Group 1 Gene1   8.087463
3 Sample 3 Group 1 Gene1   9.885696
4 Sample 4 Group 1 Gene1   7.832890
5 Sample 5 Group 1 Gene1  10.239599

> gene.exprs.long[1:5, 1:2] # print the first 5 rows and columns 1 & 2
        ID   Group
1 Sample 1 Group 1
2 Sample 2 Group 1
3 Sample 3 Group 1
4 Sample 4 Group 1
5 Sample 5 Group 1

• Usage: subset(dataset, logical conditions)

> subset(gene.exprs.long, ID == 'Sample 1') # All Sample 1 measures
          ID   Group  Gene Expression
1   Sample 1 Group 1 Gene1   9.695228
51  Sample 1 Group 1 Gene2   4.694323
101 Sample 1 Group 1 Gene3   3.733354
151 Sample 1 Group 1 Gene4   4.874305

> subset(gene.exprs.long, Expression > 10) # gene expression > 10
          ID   Group  Gene Expression
5   Sample 5 Group 1 Gene1   10.23960
6   Sample 6 Group 1 Gene1   11.01402
10 Sample 10 Group 1 Gene1   10.10460
13 Sample 13 Group 2 Gene1   10.21796
16 Sample 16 Group 2 Gene1   10.03342
46 Sample 46 Group 5 Gene1   10.11114

• This can be useful for stats when you are just starting out:

> gene1 <- subset(gene.exprs.long, Gene == 'Gene1') # only data for gene 1
> summary(aov(Expression ~ Group, data = gene1)) # run an ANOVA on it
            Df Sum Sq Mean Sq F value  Pr(>F)   
Group        4  13.08   3.271   4.879 0.00236 **
Residuals   45  30.17   0.670                   
---
Signif. codes:  0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

• Data analysts have come up with ways to describe the format of the data they work in
• Most of the data you come into contact with will be in either wide or long format
• See data files in example zip folder for wide and long versions of same data set
• The reshape2 package is good for going from wide to long and back
  • here's a good tutorial
• Keeping your data in long format will make your life in R easier!

There are three main graphing frameworks available in R for creating high quality plots:

1. Base Graphics
2. Lattice Graphics
3. ggplot2 Graphics

ggplot2 allows intro R users to create high quality data visualizations with little effort using an intuitive plotting syntax:

ggplot2 is a plotting system for R, based on the grammar of graphics, which tries to take the good parts of base and lattice graphics and none of the bad parts. It takes care of many of the fiddly details that make plotting a hassle (like drawing legends) as well as providing a powerful model of graphics that makes it easy to produce complex multi-layered graphics (taken from ggplot2 site).

• Nice walkthrough of using qplot to make complex graphics
• Excellent quick overview of ggplot2
• In depth guide of ggplot2
• Quick reference for ggplot2

• qplot() is the easiest way to get started with ggplot2
• If you haven't done so already, please run the following commands:

# Install only once.
install.packages("ggplot2")

# Load the package every time you want to use it.
library(ggplot2)

• Let's plot our gene expression data set!
• First, a basic qplot call:

qplot(x = Gene, y = Expression, data = gene.exprs.long)

• qplot guesses what the best graphical display would be given your data
• You can override this to get other graphics
  • Done by specifying the geom option

qplot(x = Gene, y = Expression, data = gene.exprs.long, geom = 'boxplot')

• The color option adds color!

qplot(x = Gene, y = Expression, data = gene.exprs.long, color = Gene)

• Or fill for some geoms

qplot(x = Gene, y = Expression, data = gene.exprs.long, geom = 'boxplot', 
      fill = Gene)

qplot(x = Group, y = Expression, data = gene.exprs.long, fill = Group, 
      facets = ~ Gene, geom = 'boxplot')

qplot(x = Group, y = Expression, data = gene.exprs.long, color = Group, 
    facets = ~Gene, geom = "jitter") + theme_bw() + 
    scale_color_brewer(type = 'qual', palette = "Dark2") + 
    theme(axis.text.x = element_text(angle = 45, hjust = 1))

• Tab-delimited files are easily exported from Excel
• One of the best ways to store data in a flat text file
• Typically have a file extension of .txt or .tsv.

ID  Group   Gene1   Gene2   Gene3   Gene4
Sample 1    Group 1 9.695   4.694   3.733   4.874
Sample 2    Group 1 8.087   3.276   2.220   3.095
Sample 3    Group 1 9.885   6.297   5.842   8.233
Sample 4    Group 1 7.832   -1.286  2.594   -1.089
Sample 5    Group 1 10.239  4.474   3.300   3.377

File -> Save As -> Tab-Delimited Text

• It is also very easy to export comma-delimited files from Excel
• Comma-delimited files typically have a file extension of .csv.

"ID","Group","Gene1","Gene2","Gene3","Gene4"
"Sample 1","Group 1",9.695,4.694,3.733,4.874
"Sample 2","Group 1",8.087,3.276,2.220,3.095
"Sample 3","Group 1",9.885,6.297,5.842,8.233
"Sample 4","Group 1",7.832,-1.286,2.594,-1.089
"Sample 5","Group 1",10.239,4.474,3.300,3.377

File -> Save As -> Comma Separated Values